KDR peptides and vaccines comprising the same

ABSTRACT

The present invention provides nonapeptides selected from peptides comprising the amino acid sequence of SEQ ID NO:2, 3, 5, 8, 11, or 12; nonapeptides or decapeptides selected from peptides comprising the amino acid sequence of SEQ ID NO:29, 30, 33, 34, 40, or 46; and peptides with cytotoxic T cell inducibility, in which one, two, or several amino acids are substituted or added to the above-mentioned amino acid sequences, as well as pharmaceuticals for treating or preventing tumors, where the pharmaceuticals comprise these peptides. The peptides of this invention can be used as vaccines.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/527,496, filed on Feb. 13, 2006, which is the National Stage Entryunder 35 U.S.C. §371 of International Application No. PCT/JP03/11722,filed Sep. 12, 2003, which claims the benefit of JP Application No.2003-167042, filed Jun. 11, 2003, JP Application No. 2003-62003, filedMar. 7, 2003 and JP Application No. 2002-267285, filed Sep. 12, 2002.The entire disclosures of each of these applications is herebyincorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

NOT APPLICABLE

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

NOT APPLICABLE

TECHNICAL FIELD

The present invention relates to novel peptides that are extremelyuseful as cancer vaccines, and pharmaceuticals that comprise thesepeptides, for treating and preventing tumors.

BACKGROUND ART

Tumor-rejection antigen genes have mainly been identified for malignantmelanomas, and accordingly, cancer immunotherapies utilizing these genesare developed. Specifically, with recognition of the importance ofCD8-positive T cells in anti-tumor immune responses, cancer vaccinetherapy that induces tumor-specific CD8-positive T cells in vivo hasreceived attention, and is being applied to various clinicalapplications. In addition, the mechanism by which peptides consisting ofapproximately ten amino acid residues activate T cells via the Class Ipathway, by the assistance of various costimulatory molecules, to inducetumor-specific cytotoxic T cells (CTLs) was also elucidated.Furthermore, peptides restricted to individual HLA molecules are beingactively identified.

However, complete control of tumors is currently impossible. This may bedue to tumor cell heterogeneity, a reduction or disappearance ofMHC-Class I expression in tumor cells, and the absence of targetmolecules in the tumor cells. Furthermore, the currently identifiedtumor antigen peptides exist in some types of tumors, but can not existin all tumor types. Thus, to resolve these problems, the presentinventors did not use tumor cells as target cells, but rather focused onthe endothelial cells of tumor vessels. More specifically, endothelialcells have hardly any problems involving a decrease or disappearance ofMHC-Class I expression, or heterogeneity. Thus, if CTLs that targettumor vessels can be induced, problems in conventional cancer vaccinetherapy, such as the disappearance of Class I and absence of a targetmolecule, may be overcome, regardless of the type of tumor, andexcellent therapeutic effects can be anticipated. Studies on tumorangiogenesis were started from a pioneering hypothesis proposed byFolkman et al. in the 1970's, and have been conducted from variousangles. Many studies have been carried out on vascular endothelialgrowth factor (VEGF)—VEGF receptors (VEGFRs) to evaluate theirsignificance in tumor angiogenesis. Angiogenesis inhibitors have beenvigorously developed as target-oriented drugs, particularly in cancertherapy, and are already being clinically tested. However, therapiesthat use this concept for cancer vaccine treatments are not yet in use.One of the reasons may be immunotolerance to VEGFR, which is expressedin normal cells. However, in the 1990's, Plate, Millauer, and Risau etal. confirmed that VEGFRs were strongly expressed in the endothelialcells of tumoral tissues. In addition, the immune response toautoantigens such as CEA and HER/neu, which are also expressed in normalcells, is not necessarily one of immunotolerance. Thus, the presentinventors reasoned that VEGFRs may be used as targets for cancer vaccinetherapy.

Recently, it was reported that active immunization against VEGFRs caninhibit angiogenesis in tumors, and metastasis (The Journal ofExperimental Medicine, 2002, 195:12, 1575-1584). However, thisliterature merely used soluble VEGFR proteins, and made no investigationof the amino acid sequences of effective peptides.

SUMMARY OF THE INVENTION

The present inventors focused on possible cancer vaccine therapies thattarget VEGFR2 (KDR/flk-1; referred to below as KDR), where VEGFR2 isstrongly expressed in tumoral tissue endothelial cells, and is thoughtbe involved in the proliferation of endothelial cells on the VEGFsignal. Furthermore, the present inventors screened for peptides thatcan be effectively used as vaccines, examining their specificity, andcompleting this invention.

More specifically, the present invention provides [1] to [22] asfollows.

[1] A nonapeptide selected from a group of peptides comprising the aminoacid sequence of SEQ ID NO:2, 3, 5, 8, 11, or 12.

[2] A peptide with cytotoxic T cell inducibility, wherein one, two, ormore amino acids are substituted or added to the amino acid sequence ofSEQ ID NO:2, 3, 5, 8, 11, or 12.

[3] The peptide of [2], wherein the second amino acid from the Nterminus is phenylalanine, tyrosine, methionine, or tryptophan.

[4] The peptide of [2] or [3], wherein the C-terminal amino acid isphenylalanine, leucine, isoleucine, tryptophan, or methionine.

[5] A nonapeptide or decapeptide selected from a group of peptidescomprising the amino acid sequence of SEQ ID NO:29, 30, 33, 34, 40, or46.

[6] A peptide with cytotoxic T cell inducibility, wherein one, two, ormore amino acids are substituted or added to the amino acid sequence ofSEQ ID NO:29, 30, 33, 34, 40, or 46.

[7] The peptide of [6], wherein the second amino acid from the Nterminus is leucine or methionine.

[8] The peptide of [6] or [7], wherein the C-terminal amino acid isvaline or leucine.

[9] A pharmaceutical for treating and/or preventing tumors, wherein thepharmaceutical comprises one or more peptides of any one of [1] to [8].

[10] A pharmaceutical for treating diabetic retinopathy, chronicrheumatoid arthritis, psoriasis, and atherosclerosis, wherein thepharmaceutical comprises one or more peptides of any one of [1] to [8].

[11] An exosome that presents on its surface a complex comprising apeptide of any one of [1] to [8], and an HLA antigen.

[12] The exosome of [11], wherein the HLA antigen is HLA-A24 or HLA-A02.

[13] The exosome of [12], wherein the HLA antigen is HLA-A2402 orHLA-0201.

[14] A method for inducing an antigen-presenting cell with highcytotoxic T cell inducibility by using a peptide of any one of [1] to[8].

[15] A method for inducing a cytotoxic T cell by using a peptide of anyone of [1] to [8].

[16] A method for inducing an antigen-presenting cell with highcytotoxic T cell inducibility, wherein said method comprises the step ofintroducing a gene that comprises a polynucleotide encoding a peptide ofany one of [1] to [8] into an antigen-presenting cell.

[17] An isolated cytotoxic T cell that is induced by using a peptide ofany one of [1] to [8].

[18] An antigen-presenting cell that presents a complex of an HLAantigen and a peptide of any one of [1] to [8].

[19] The antigen-presenting cell of [18], which is induced by the methodof [14] or [15].

[20] A vaccine for inhibiting angiogenesis at a diseased site, whereinthe vaccine comprises a peptide of any one of [1] to [8] as an activeingredient.

[21] The vaccine of [20], which is used for administration to a subjectwhose HLA antigen is HLA-A24 or HLA-A02.

[22] The vaccine of [20] or [21], which is used to suppress the growthand/or metastasis of malignant tumors.

The present specification comprises the contents described in thespecifications and/or drawings of Japanese Patent Application Nos.2002-267285, 2003-062003, and 2003-167042, on which the priority rightsof this invention are based.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cytotoxicity of CTL clones against the target cells.

FIG. 2 shows the results of HLA-tetramer analyses of established CTLclones:

(a) CTL clones induced by the peptides of this invention, and feedercells; and

(b) feeder cells (control).

FIG. 3 shows the effect of vaccination using the peptides of thisinvention on the survival rate of Colon 26-inoculated BALB/c mice.

FIG. 4 shows the effect of vaccination using the peptides of thisinvention on the growth of Colon 26-derived tumors.

FIG. 5 shows the effect of vaccination using the peptides of thisinvention on the survival rate of B16-inoculated mice.

FIG. 6 shows the effect of vaccination using the peptides of thisinvention on the growth of B16 melanoma-derived tumors. Before challengewith a subcutaneous injection of B16 tumor cells, A2/Kb TGM werevaccinated twice, with a one-week interval, with DC pulsed with KDR773(black circles), DC alone (white triangles), or HBSS (white squares).The figure shows mean tumor growth (P<0.01).

FIG. 7 shows the cytotoxic effect of CTL clone C29-169 onHLA-A24-positive cells presenting peptides of this invention.

FIG. 8 shows the cytotoxic effect of CTL clone C29-169 onHLA-A24-positive cells presenting peptides of this invention.

FIG. 9 shows the cytotoxic effect of CTL clone KDR C85-775 (KWC85-775)on HLA-A0201-positive cells presenting peptides of this invention.

FIG. 10 shows the cytotoxic effect of CTL clone KDR C85-775 (KWC85-775)on HLA-A0201-positive cells presenting KDR peptides.

FIG. 11 shows the cytotoxic effect of CTL clone KDR C51-1328 (KWC51) onHLA-A0201-positive cells presenting peptides of this invention.

FIG. 12 shows the cytotoxic effect of CTL clone KDR C51-1328 (KWC51) onHLA-A0201-positive cells presenting KDR peptides.

FIG. 13 shows the cytotoxic effect of CTL clone KDR C44-773-2L(KWC44-773-2L) on HLA-A0201-positive cells presenting peptides of thisinvention.

FIG. 14 shows the cytotoxic effect of CTL clone KDR C44-773-2L(KWC44-773-2L) on HLA-A0201-positive cells presenting peptides of thisinvention.

FIG. 15 shows the cytotoxic effect of CTL clone KDR C44-773-2L(KWC44-773-2L) on HLA-A0201-positive cells presenting KDR peptides.

FIG. 16 shows the inhibitory effect of each type of antibody on thecytotoxic effect of the CTL clones.

FIG. 17 shows the inhibitory activity of the peptides of this inventionon the B16 cell-induced angiogenic response in A2/Kb TGM. Mice werevaccinated twice with HBSS, unpulsed DC, or DC pulsed with the epitopepeptides of this invention (KDR190, KDR772, KDR773, KDR773-2L, KDR775,or KDR1084). The arrows indicate newly formed blood vessels, which runin a characteristic zigzag pattern.

FIG. 18 shows the suppressive effect of the peptides of this inventionon the increase in volume of human tumors in transgenic mice.

FIG. 19 shows the cytotoxic effect of CTL clone KWC65-190 onHLA-A0201-positive cells presenting peptides of this invention, in thepresence (black boxes) or absence (white boxes) of the peptides.

FIG. 20 shows the cytotoxic effect of CTL clone KWC72-772 onHLA-A0201-positive cells presenting peptides of this invention, in thepresence (black boxes) or absence (white boxes) of the peptides.

FIG. 21 shows the cytotoxic effect of CTL clone KWC72-772 onHLA-A0201-positive cells presenting KDR peptides. HePG2-VEGFR2: blackboxes; HePG2-EGFP: white boxes.

FIG. 22 shows the cytotoxic effect of CTL clone C7-1318 onHLA-A24-positive cells presenting peptides of this invention, in thepresence (black boxes) or absence (white boxes) of the peptides.

FIG. 23 shows the cytotoxic effect of CTL clone KWC46 onHLA-A24-positive cells presenting peptides of this invention, in thepresence (black boxes) or absence (white boxes) of the peptides.

FIG. 24 shows the cytotoxic effect of CTL clone C18-189 onHLA-A24-positive cells presenting peptides of this invention, in thepresence (black boxes) or absence (white boxes) of the peptides.

FIG. 25 shows the cytotoxic effect of CTL clone C65-826 onHLA-A24-positive cells presenting peptides of this invention, in thepresence (black boxes) or absence (white boxes) of the peptides.

FIG. 26 shows the cytotoxic effect of CTL clone C7-1318 onHLA-A24-positive cells presenting peptides of this invention.HT29-VEGFR2: black boxes; HT29-EGFP: white boxes.

FIG. 27 shows the cytotoxic effect of CTL clone KWC46 onHLA-A24-positive cells presenting peptides of this invention.HT29-VEGFR2: black boxes; HT29-EGFP: white boxes.

FIG. 28 shows the cytotoxic effect of CTL clone C18-189 onHLA-A24-positive cells presenting peptides of this invention.HT29-VEGFR2: black boxes; HT29-EGFP: white boxes.

FIG. 29 shows the cytotoxic effect of CTL clone C65-826 onHLA-A24-positive cells presenting peptides of this invention.HT29-VEGFR2: black boxes; HT29-EGFP: white boxes.

FIG. 30 shows the inhibitory activity of the peptides of this inventionon the Colon 26 cell-induced angiogenic response in BALB/c mice. Themice were vaccinated twice with HBSS, unpulsed DC, or DC pulsed with anepitope peptide of this invention (KDR189 or KDR826). The arrowsindicate newly formed blood vessels, which run in a characteristiczigzag pattern.

FIG. 31 shows the inhibition of Colon 26 cell-induced angiogenicresponse in BALB/c by the peptides of this invention. The bars indicatethe average, and the vertical error bars indicate standard error.

FIG. 32 shows the cytotoxic effect of CTL, derived from patientsstimulated with the peptides of this invention, on HLA-A24-positivecells, in the presence (black diamonds) or absence (white squares) ofthe peptides.

(A) The cytotoxic effect of colon cancer patient-derived CTL obtained bystimulation with SEQ ID NO:8 (in which the amino acid initiationposition is KDR169);

(B) The cytotoxic effect of colon cancer patient-derived CTL obtained bystimulation with SEQ ID NO:5 (in which the amino acid initiationposition is KDR189); and

(C) The cytotoxic effect of colon cancer patient-derived CTL obtained bystimulation with SEQ ID NO:3 (in which the amino acid initiationposition is KDR220).

DETAILED DESCRIPTION OF THE INVENTION Mode for Carrying Out theInvention

The present inventors first considered that various proteins arepresented in vivo on antigen presenting cells after being degraded to9-mer peptides (nonapeptides), and examined the binding affinity of9-mer or 10-mer partial peptides of KDR proteins to HLA antigens, whichare human major histocompatibility antigens (MHC antigens). The lowercase letters in the peptide sequences shown on the right of Table 1indicate the fifth amino acids.

The amino acid sequence of human KDR protein is well known, disclosed byU.S. Pat. No. 5,861,301, for example, and can be easily obtained by oneskilled in the art. The 9-mer and 10-mer peptides can be obtained bysynthesizing peptides initiated from any position, based on thefull-length amino acid sequence of the obtained KDR protein. Peptidescan be synthesized according to conventional methods used in peptidechemistry. Commonly used synthesis methods are described, for example,in literatures such as Peptide Synthesis, Interscience, New York, 1966;The Proteins, vol. 2, Academic Press Inc., New York, 1976; PeptideSynthesis (Peputido Gousei), Maruzen Co., Ltd., 1975; Foundations andExperiments in Peptide Synthesis (Peputido Gousei no Kiso to Jikken),Maruzen Co., Ltd., 1985; and Development of Pharmaceuticals, New Series(Tyakuhin no Kaihatsu, Zoku), Volume 14, Peptide Synthesis (PeputidoGousei), Hirokawa Shoten, 1991, and in publications such asInternational Publication No. WO 99/67288. Binding with HLA antigens canbe measured by isolating cells that comprise HLA antigens on their cellsurface, such as dendritic cells, and using conventional methods tomeasure the binding of peptides to cells.

Alternatively, software programs now available on the Internet, such asthose described in Parker, K. C., J. Immunol., 152, 1994, may be used tocalculate the binding affinities between various peptides and HLAantigens in silico. Binding affinity with HLA antigens can be measuredas described, for example, in Parker, K. C., J. Immunol., 152, 1994; andNukaya, I., Int. J. Cancer, 80, 1999.

To obtain sufficient results, A-24 type and A-02 type antigens, said tobe highly expressed among the Japanese, are preferably used as the HLAantigens. More preferably, subtypes such as A-2402 and A-0201 are used.However, clinically, by predetermining the HLA antigen type of a patientin need of treatment, a peptide can be appropriately selected to have ahigh level of binding affinity to that antigen, or level of cytotoxic Tcell (CTL) inducibility upon antigen presentation. Furthermore, toobtain peptides with high levels of binding affinity and CTLinducibility, substitution or addition of one, two, or several aminoacids may be performed based on the amino acid sequence of the naturallyoccurring KDR partial peptide. Herein, the term “several” means five orless, or preferably three or less. In addition to peptides in nature,the sequence regularity of the peptides displayed by binding to HLAantigens is already known (J. Immunol., 152:3913, 1994; Immunogenetics,41:178, 1995; J. Immunol., 155:4307, 1994). Thus, modifications based onthis regularity can also be performed on the obtained peptides. Forexample, peptides in which the second amino acid from the N terminus issubstituted with phenylalanine, tyrosine, methionine, or tryptophan, andin which the C terminal amino acid is substituted with phenylalanine,leucine, isoleucine, tryptophan, or methionine may also be preferablyused as peptides with high HLA-24 binding affinity. On the other hand,peptides in which the second amino acid from the N terminus issubstituted with leucine or methionine, and in which C terminal aminoacid is substituted with valine or leucine may be preferably used aspeptides with high HLA-0201 binding affinity. Furthermore, one to twoamino acids may also be added to the N and/or C terminus of thepeptides.

However, when a peptide sequence is identical to a portion of an aminoacid sequence of an endogenous or exogenous protein with a differentfunction, it may cause side effects such as autoimmune diseases orallergic symptoms against specific substances. Therefore, it ispreferable to use available databases to carry out homology searches, toavoid situations in which a sequence matches the amino acid sequence ofanother protein. Furthermore, if homology searches reveal that nopeptides comprising even a one- or two-amino acid difference exist,there is no risk of such problems caused by modifications of theabove-mentioned amino acid sequences for increasing the binding affinitywith HLA antigens and/or the CTL inducibility.

As described above, peptides with high binding affinity to the HLAantigens are expected to be highly effective as cancer vaccines.However, it is necessary to determine whether the candidate peptides,which are selected using high binding affinity as an indicator, actuallyhave CTL inducibility or not. Confirmation of CTL inducibility iscarried out by inducing antigen-presenting cells comprising human MHCantigens (such as B-lymphocytes, macrophages, and dendritic cells), morespecifically dendritic cells derived from human peripheral bloodmononuclear leukocytes; stimulating the cells with the peptides; mixingthe cells with CD8-positive cells; and then measuring cytotoxicityagainst the target cells. As the reaction system, transgenic animalsgenerated to express a human HLA antigen may be used (for example, thosedescribed in Hum. Immunol. 2000 August; 61 (8):764-79 Related Articles,Books, Linkout Induction of CTL response by a minimal epitope vaccine inHLA A*0201/DR1 transgenic mice: dependence on HLA class II restrictedT(H) response, BenMohamed, L., Krishnan, R., Longmate, J., Auge, C.,Low, L., Primus, J., and Diamond, D. J.). For example, by radiolabelingthe target cells with ⁵¹Cr or such, cytotoxicity can be calculated fromthe radioactivity released from the target cells. Alternatively, theactivity can be examined by measuring the IFN-γ produced and released byCTL in the presence of antigen-presenting cells that have immobilizedpeptides, and visualizing the inhibition zone on the media usinganti-IFN-γ monoclonal antibodies.

The results of examining the CTL inducibility of peptides as describedabove revealed that those with high binding affinity to HLA antigens donot necessarily have high inducibility. Furthermore, nonapeptidesselected from peptides comprising the amino acid sequences of VYSSEEAEL(SEQ ID NO:2), GYRIYDVVL (SEQ ID NO:3), SYMISYAGM (SEQ ID NO:5),RFVPDGNRI (SEQ ID NO:8), KWEFPRDRL (SEQ ID NO:11), or DFLTLEHLI (SEQ IDNO:12), and nonapeptides and decapeptides selected from peptidescomprising the amino acid sequence of AMFFWLLLV (SEQ ID NO:29),VIAMFFWLL (SEQ ID NO:30), AVIAMFFWL (SEQ ID NO:33), KLIEIGVQT (SEQ IDNO:34), YMISYAGMV (SEQ ID NO:40), or IQSDVWSFGV (SEQ ID NO:46), showedparticularly high CTL inducibility.

The present invention further provides peptides with cytotoxic T cellinducibility, wherein one, two, or several amino acids are substitutedwith or added to the amino acid sequence of SEQ ID NO:2, 3, 5, 8, 11, or12. One, two, or several amino acids can be substituted or added to theamino acid sequences of SEQ ID NOs:2, 3, 5, 8, 11, and 12, consisting ofnine amino acids, as long as they have CTL inducibility and do not matchthe amino acid sequence of another protein. In particular, for example,the second amino acid from the N terminus is preferably substituted tophenylalanine, tyrosine, methionine, or tryptophan, or the C terminalamino acid is preferably substituted to phenylalanine, leucine,isoleucine, tryptophan, or methionine; or one or two amino acids areadded to the N terminus and/or C terminus.

The present invention also provides peptides with cytotoxic T cellinducibility, wherein one, two, or several amino acids are substitutedor added to the amino acid sequence of SEQ ID NO:29, 30, 33, 34, 40, or46. One, two, or several amino acids can be substituted or added to theamino acid sequences of SEQ ID NOs:29, 30, 33, 34, 40, and 46,consisting of nine or ten amino acids, as long as they have CTLinducibility and do not match the amino acid sequence of anotherprotein. In particular, for example, the second amino acid from the Nterminus is preferably substituted to leucine or methionine, or the Cterminal amino acid is preferably substituted to valine or leucine; orone or two amino acids are added to the N terminus and/or C terminus. Anexample of such a modified peptide is the peptide of SEQ ID NO:30, inwhich the second amino acid from the N terminus is substituted toleucine (SEQ ID NO:54), but this example is not limiting. CTL clonesobtained on stimulation with these modified peptides can recognize theoriginal peptides, and cause damage.

The peptides of this invention can be used alone or in combinations oftwo or more, as cancer vaccines capable of inducing CTL in vivo. Byadministering the peptides of this invention, the peptides are presentedat a high density on the HLA antigens of antigen-presenting cells. CTLsthat specifically react with the complexes formed between the displayedpeptides and the HLA antigens are induced, increasing aggression againstvascular endothelial cells in the tumor cells to be targeted.Alternatively, antigen-presenting cells on whose cell surface thepeptides of this invention are immobilized can be obtained by derivingdendritic cells from a subject, and stimulating them with the peptidesof this invention. The obtained antigen-presenting cells arere-administered to the subjects to induce CTL in the subjects. As aresult, aggression towards the target cells can be increased.

More specifically, the present invention provides pharmaceuticals fortreating tumors or preventing proliferation, metastasis, and such oftumors, where the pharmaceuticals comprise one or more peptides of thisinvention. Angiogenesis at pathologic sites is closely associated withnot only tumors, but also with diseases such as diabetic retinopathy,chronic rheumatoid arthritis, psoriasis, and atherosclerosis, and themetastasis of solid tumors (Folkman, J., Nature Med., 1:27-31 (1995);Bicknell, R. and Harris, A. L., Curr. Opin. Oncol., 8:60-65 (1996)).Therefore, the peptides of this invention can be used to treat tumors,diseases such as diabetic retinopathy, chronic rheumatoid arthritis,psoriasis, atherosclerosis, and metastasis of solid tumors.

The peptides of this invention were confirmed to inhibit the formationof tortuous blood vessels, which are morphologically different fromnormal blood vessels, and which are formed in malignant tumoral tissues.The results of analyzing wound healing and fertility in vaccinated micealso confirmed that the peptides do not have an adverse effect on normalphysiological angiogenesis. Furthermore, by using CTL clones thatrecognize the peptides of this invention, cytotoxicity againstnon-proliferative or proliferative endothelial cells was tested invitro. These clones showed stronger activity towards proliferativeendothelial cells than non-proliferative endothelial cells. Morespecifically, they can function very specifically for disordersinvolving proliferative endothelial cells, and particularly cancer.

In vivo and in vitro stimulation of dendritic cells by the peptides ofthis invention can be easily performed by exposing the cells to a highconcentration of the peptides, which causes these peptides to replacethe peptides originally immobilized on the cells. Therefore, thepeptides used in this invention must have at least a certain level ofbinding affinity to HLA antigens.

The pharmaceuticals of this invention may be directly administered asthe peptides of this invention themselves, or may be administered aspharmaceutical compositions that have been formulated by conventionalformulation methods. In such cases, the pharmaceuticals of thisinvention can appropriately include, in addition to the peptides of thisinvention, carriers, excipients, and such that are ordinarily used forpharmaceuticals, without particular limitations. The pharmaceuticals ofthis invention can be used for treatment and prevention of varioustumors, such as gastric cancer, duodenal cancer, colon cancer, lungcancer, breast cancer, prostate cancer, and brain tumors. The peptidesof this invention do not target the tumor cells themselves, but targetthe endothelial cells of blood vessels that are newly formed in tumoraltissues. Therefore, a wide variety of tumors can be treatment targets,and the pharmaceuticals of this invention are not particularly limitedin their use.

Pharmaceuticals for treating and/or preventing tumors, which comprise apeptide of this invention as an active ingredient, can be administeredwith adjuvants so that cellular immunity is induced effectively; can beadministered with other active ingredients such as antitumor agents; andcan be administered in granular forms. Those adjuvants described in theliterature (Clin. Microbiol. Rev., 7:277-289, 1994) or such areapplicable. Furthermore, the pharmaceuticals of this invention can beadministered as liposome formulations, as granular formulations bound tobeads of a few micrometers in diameter, and as formulations to whichlipids are bound. Administration methods may be carried out, forexample, orally, intradermally, or subcutaneously, or throughintravenous injection, or such. Systemic administration or localadministration to the vicinity of the target tumor may be applicable.Doses of the peptides of this invention can be adjusted appropriately,depending on the disease to be treated, age and weight of the patients,administration methods, and such. Ordinarily, 0.001 mg to 1,000 mg,preferably 0.01 mg to 100 mg, more preferably 0.1 mg to 10 mg, of thepeptides of this invention are preferably administered once in a fewdays to a few months. One skilled in the art can appropriately selectsuitable doses.

Alternatively, the present invention provides intracellular vesicleswhich present complexes formed between the peptides of this inventionand HLA antigens on their surface. These intracellular vesicles arecalled exosomes. Exosomes can be prepared, for example, according to themethods specifically described in Published Japanese Translation ofInternational Publication Nos. Hei 11-510507 and 2000-512161. Exosomescan preferably be prepared using antigen-presenting cells obtained fromsubjects who are to be the target of therapy or prophylaxis. Theexosomes of this invention can be inoculated as cancer vaccines, as forthe peptides of this invention.

The type of HLA antigens to be used must match that of the subject inneed of therapy and/or prophylaxis. For example, for Japanese people,HLA-A24 or HLA-A02, particularly HLA-A2402 or HLA-0201, is oftenappropriate.

The present invention also provides methods for inducingantigen-presenting cells using the peptides of this invention. Theantigen-presenting cells can be induced by inducing dendritic cells fromperipheral blood monocytes; and then contacting (stimulating) them withthe peptides of this invention, in vitro or in vivo. Administering thepeptides of this invention to subjects induces in the body of thesubject antigen-presenting cells to which the peptides of this inventionare immobilized. Alternatively, the peptides of this invention can beimmobilized to the antigen-presenting cells to be administered to thesubject as a vaccine.

The present invention also provides methods for inducingantigen-presenting cells with a high level of cytotoxic T cellinducibility, wherein said methods comprise the in vitro introduction toan antigen-presenting cell of a gene that comprises a polynucleotideencoding a peptide of this invention. The genes to be introduced may bein the form of DNA or RNA. The introduction methods may be variousmethods commonly used in the art, such as lipofection, electroporation,and calcium phosphate methods, without particular limitations. Morespecifically, the methods may be performed as described in, for example.Cancer Res., 56:5672, 1996; J. Immunol., 161:5607, 1998; J. Exp. Med.,184:465, 1996; or Published Japanese Translation of InternationalPublication No. 2000-509281. By introducing the genes intoantigen-presenting cells, the genes undergo transcription, translation,and such in the cells. The obtained proteins are then subjected to ClassI or II MHC processing and a presentation pathway to present partialpeptides.

Furthermore, the present invention provides methods for inducing CTLsusing the peptides of this invention. By administering the peptides ofthis invention to a subject, CTLs are induced in the body of thesubject, enhancing immunity against the angiogenic endothelial cells inthe tumoral tissues. Alternatively, the methods may be used for ex vivotherapeutic methods, in which subject-derived, antigen-presenting cells,CD8-positive cells, or peripheral blood mononuclear leukocytes arecontacted (stimulated) in vitro with the peptides of this invention toinduce CTLs, and then the cells are returned to the subject.

In addition, the present invention provides cytotoxic T cells, that areinduced using the peptides of this invention and then isolated. Thecytotoxic T cells, which have been induced by stimulation withantigen-presenting cells that present the peptides of this invention,are preferably derived from subjects to be the target of therapy and/orprophylaxis. The cytotoxic T cells can be administered alone or, for thepurpose of antitumor effect, in combination with other drugs, includingthe peptides, exosomes and so on of this invention. The obtainedcytotoxic T cells act specifically against target cells presenting thepeptides of this invention, or preferably, against target cellspresenting the same peptides used for induction. The target cells may becells that endogenously express KDR, or cells forced to express KDR.Furthermore, cells that present the peptides of this invention on theircell surface due to stimulation by these peptides can also be targeted.

The present invention also provides antigen-presenting cells thatpresent complexes formed between HLA antigens and the peptides of thisinvention. The antigen-presenting cells that are obtained by contactwith the peptides of this invention, or with nucleotides encoding thepeptides of this invention, are preferably derived from subjects to betargeted for therapy and/or prophylaxis. The antigen-presenting cellscan be administered as vaccines alone, or in combination with otherdrugs such as the peptides of this invention, exosomes, and cytotoxic Tcells.

BEST MODE FOR CARRYING OUT THE INVENTION

Herein below, the present invention will be specifically described usingExamples, but it is not to be construed as being limited thereto.

Example 1 Prediction of VEGFR-2 (KDR)-Derived Peptides-1 (HLA-A*2402)

Based on the entire amino acid sequence of KDR protein, twelve types of9-mer peptides (SEQ ID NOs:1 to 12), and twelve types of 10-mer peptides(SEQ ID NOs:13 to 24) were predicted in order from that with the highestbinding affinity to HLA-A*2402, using BioInformatics & MolecularAnalysis Section (BIMAS) HLA Peptide Binding Prediction software (on theWorld Wide Web atbimas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform) (Table 1). Table1 shows the binding affinity of each of the 9 mers and 10 mers in orderfrom the highest value, together with the position of their N termini inthe amino acid sequence of the KDR protein. In the table, CE652 (SEQ IDNO:25) refers to one of the epitopes of tumor antigen CEA(carcinoembryonic antigen) that has been reported by Nukaya, I. (Int. J.Cancer, 80, 1999). HIV peptides (ILKEPVHGV (SEQ ID NO:55) and RYLRDQQLL(SEQ ID NO:56)) were used as the negative control peptides.

The peptides were synthesized according to standard solid-phasesynthesis methods using Cleaved PepSet (Mimotope, San Diego, Calif.),and purified by reverse phase HPLC. The purity (>95%) and the type ofpeptide were individually determined using HPLC and mass spectrometry.

TABLE 1 Binding of HLA-A*2402 to KDR-derived peptides SEQ ID INITIATIONAA SEQUENCE BINDING SEQ ID INITIATION AA SEQUENCE BINDING NO: POSITION(9 mers) AFFINITY*¹ NO: POSITION (10 mers) AFFINITY*¹ 1 KDR583 WYKLGPQPL240 13 KDR950 DYVGaIPVDL 420 2 KDR1318 VYSSEEAEL 220 14 KDR434SYQYgTTQTL 360 3 KDR220 GYRIYDVVL 200 15 KDR1058 DYVRkGDARL 300 4 KDR920KFGNLSTYL 48 16 KDR304 LYTCaASSGL 200 5 KDR189 SYMISYAGM 38 17 KDR1318VYSSeEAELL 200 6 KDR828 EFPRDRLKL 33 18 KDR734 LYTCqACSVL 200 7 KDR1152TFSELVEHL 29 19 KDR926 TYLRsKRNEF 198 8 KDR169 RFVPDGNRI 22 20 KDR353KYLGyPPPEI 165 9 KDR331 AFGSGMESL 20 21 KDR116 VYVQdYRSPF 150 10 KDR604KNLDTLWKL 16 22 KDR395 NYTViLTNPI 72 11 KDR826 KWEFPRDRL 12 23 KDR777FFWLILVIIL 24 12 KDR998 DFLTLEHLI 9 24 KDR193 SYAGmVFCEA 9.2 25 CE652*²TYACFVSNL 200 *¹Parker KC: 1994 J.Immunol 152 *²Nukaya I: 1999Int.J.Cancer 80

Example 2 Establishment of CTL Lines Using the Predicted Peptides

Epstein-Barr virus (EBV)-immortalized B cell lines, TISI (HLA-A24/24)and EHM (HLA-A3/3), were provided by Takara Shuzo Biotechnology ResearchLaboratory.

HLA serotyping was performed using 7 mL of peripheral blood collectedfrom a healthy subject. Peripheral blood mononuclear lymphocytes (PBMCs)were isolated from HLA-A24-positive peripheral blood using Ficoll Paque(Pharmacia) specific gravity centrifugation.

The obtained PBMCs were left to stand for ten hours in a culture flask(Corning, 43072). After removing the suspended cells, the cells thatadhered to the flask were cultured in AIM-V medium (Invitrogen)supplemented with 2% autoserum by adding 1,000 U/mL of human GM-CSF(provided by Kirin Brewery), and 1,000 U/mL of human IL-4 (Genzyme).Five days later, the cells were cultured for another 48 hours in 10μg/mL of OK-432 (provided by Chugai Pharmaceutical Co., Ltd.), and wereused as antigen-presenting cells for CTL induction. Dendritic cells(DCs) were confirmed to be DCs by reaction with FITC-labeledanti-ClassII, CD80, and CD86 antibodies, and with PE-labeledanti-ClassI, CD11c, CD40 (all from Beckton-Dickinson), and CD83(Immunotech) antibodies; followed by analysis of surface antigens byFACS-Calibur (Beckton-Dickinson) using Cell Quest software.

Pre-induced DCs were pulsed for four hours at 20° C. with twelve kindsof 9-mer peptides (SEQ ID NOs:1 to 12) (20 μg/mL), that were predictedin Example 1 to have high binding affinity, in the presence of 3 μg/mLof β2-microglobulin. The resulting DCs were mixed with CD8-positivecells selected by magnetic beads (Dynabeads M-450 and Detachabeads) fromPBMC, at a ratio of 1:20 or 1:2, and then cultured in a 48-well plate(Corning), in the presence of 10 ng/mL of human IL-7 (Genzyme). Threedays later, the final concentration of 10 U/mL of IL-2 (Sigma) was addedto the culture. And seven and 14 days later, the same DCs werestimulated to induce CTLs, and 20 days later, cytotoxicity was measuredfor each well using peptide-pulsed TISI cells as targets. Only thepositive wells were cultured by a stimulation method with allo-PBMC,EBV-immortalized B cell lines (EHM), and 30 ng/mL of anti-CD3 antibody.CTLs were functionally analyzed 14 days later.

Cytotoxic activity was evaluated by a four-hour ⁵¹Cr release assay. Aconcentration of 20 μg/mL of the target cells were pulsed with peptidesovernight. The target cells were labeled with 100 μCi of Na₂ ⁵¹CrO₄ at37° C. for one hour, and then washed three times with RPMI1640. Thetarget cells (1×10⁴/100 μL) and 100 μL of effector cells at variousconcentrations with a total volume of 200 μL were placed into a U-shaped96-well microtiter plate (Corning), and cultured at 37° C. in a CO₂incubator for four hours. After culturing, 100 μL of supernatant wascollected from each well, and measured using a γ counter. Natural decaywas the radioactivity of the target cells and the medium, and maximumdecay was the radioactivity of the target cells and 1 M HCl. Cytotoxicactivity (percentage of specific lysis) was calculated by the followingformula:Cytotoxic activity(% Cytotoxicity)=(Experimental decay−naturaldecay)/(maximum decay−natural decay)×100

Six types of CTL lines were established as a result, as shown in Table2. In particular, nonapeptides shown by SEQ ID NO:2 (amino acidinitiation position KDR1318), SEQ ID NO:3 (KDR220), SEQ ID NO:5(KDR189), SEQ ID NO:8 (KDR169), SEQ ID NO:11 (KDR826), and SEQ ID NO:12(KDR998) were shown to be effective as epitope peptides.

TABLE 2 CYTOTOXIC ACTIVITY SEQ ID INITIATION BINDING ×20 ×2 NO: POSITIONAA SEQUENCE AFFINITY*¹ Pep(+) Pep(−) Pep(+) Pep(−) 1 KDR583 WYKLGPQPL240  6%  4%  0% 0% 2 KDR1318 VYSSEEAEL 220 39% 21%  8% 3% 3 KDR220GYRIYDVVL 200 57%  2% 20% 0% 4 KDR920 KFGNLSTYL 48  3%  4%  0% 2% SKDR189 SYMISYAGM 38 41%  3% 20% 0% 6 KDR828 EFPRDRLKL 33  3%  2%  1% 0%7 KDR1152 TFSELVEHL 29  0%  0%  0% 0% 8 KDR169 RFVPDGNRI 22 98%  2% 53%0% 9 KDR331 AFGSGMESL 20  6%  6%  0% 0% 10 KDR604 KNLDTLWKL 16  3%  2% 1% 1% 11 KDR826 KWEFPRDRL 12 23%  0% 11% 0% 12 KDR998 DFLTLEHLI 9 13% 4%  8% 0% 26 CE652*² TYACFVSNL 200 29% 16%  7% 1% *¹Parker.KC:1994.J.Immunol 152 *²Nukaya I: 1999 Int.J.Cancer 80

Example 3 Establishment of CTL Clones Derived from Predicted Peptides

The six types of CTL lines established in Example 2 were diluted to 0.3,1, or 3 cells/well in a U-shaped 96-well plate. 7×10⁴ cells/well ofallo-PBMC, 1×10⁴ cells/well of EHM, 30 ng/mL of anti-CD3 antibody, and125 U/mL of IL-2 were added to each well, and AIM-V supplemented with 5%autoserum was added thereto, so that total concentration was 150μL/well. Ten days later, 50 μL/well of IL-2-supplemented medium adjustedwas added to a final IL-2 concentration of 125 U/mL. CTLs werefunctionally analyzed on the 14th day, and CTLs with activity werecultured on a large scale.

As a result, from each of the five types of nonapeptides shown by SEQ IDNO:8 (amino acid initiation position KDR169), SEQ ID NO:5 (KDR189), SEQID NO:3 (KDR220), SEQ ID NO:2 (KDR1318), and SEQ ID NO:11 (KDR826),eight types (C13-169, C19-169, C29-169, C53-169, C72-169, C61-169,K5-169, and K25-169), two types (C12-189 and C18-189), eleven types(KWC3, 23, 25, 26, 36, 42, 46, 58, 61, 70, and 77), one type (C7-1318),and one type (C65-826) of CTL clones were established, respectively(Table 3).

TABLE 3 CYTOTOXIC ACTIVITY ×20 ×2 CLONE NAME Pep(+) Pep(−−) Pep(+)Pep(−−) C13-169 98% 2% 66% 2% C19-169 94% 2% 50% 0% C29-169 100%  1% 70%0% C53-169 100%  2% 91% 2% C72-169 87% 1% 39% 0% C61-169 88% 0% 38% 0%K5-169 36% 0%  6% 0% K25-169 27% 0%  5% 0% C12-189 27% 18%   7% 3%C18-189 67% 18%  28% 7% KWC3 54% 2% 11% 1% KWC23 95% 1% 49% 1% KWC25 93%1% 70% 1% KWC26 87% 1% 34% 0% KWC36 62% 2% 11% 2% KWC42 67% 0% KWC46 94%1% 51% 0% KWC58 76% 0% KWC61 70% 2% 19% 1% KWC70 92% 3% 64% 2% KWC77 99%3% 70% 2% C65-826 38% 6%  8% 3% C7-1318 84% 1% 62% 1%

Example 4 Measurement of the Cytotoxicity of Established CTL Clones

The degrees of cytotoxicity of the ten types of CTL clones establishedin Example 3 were examined by varying their ratio with the target cells(E:T ratio).

As a result, C53-169 derived from the nonapeptide of SEQ ID NO:8 (aminoacid initiation position KDR169) showed the strongest cytotoxicity.

Example 5 HLA-Tetramer Analysis of Established CTL Clones

An HLA-tetramer was synthesized from HLA-A*2402 and the peptide of SEQID NO:8 (amino acid initiation position KDR169). More specifically,plasmid vectors expressing the H chain (approximately 35 kDa) and Lchain (approximately 11 kDa) were produced individually, and theirrecombinant protein was expressed using E. coli. The H-chain plasmidvector consists only of the extracellular domain, in which the carboxylterminus was substituted with a biotinylation enzyme recognitionsequence. The H chain and L chain form an HLA peptide complex byrefolding with an antigenic peptide. The HLA peptide complexes werebiotinylated by a biotinylation enzyme, excess biotin was removed by gelfiltration chromatography and ion exchange chromatography, and thebiotinylated HLA peptide complexes were isolated and purified.HLA-tetramer was synthesized by reacting the obtained biotinylated HLApeptide complex with streptavidin (SA) at a mole ratio of 4:1 to form atetramer. When analyzing the samples, SA fluorescently labeled withphycoerythrin (PE) (SA-PE) was used to form the tetramer. CD8 moleculeis known to bind to the 245th amino acid in the α3 domain, which isalanine, with the exception of portions of HLA-A, B, and C. Therefore,the present inventors mutagenized the alanine in the HLA-tetramers tovaline, to produce a mutant HLA-tetramer capable of selectivelydetecting CTLs with high avidity towards HLA peptide complexes,independent of the binding between the CD8 molecule and the α3 domain.

Established CTL clones were detected by the synthesized HLA-A24KDR169-tetramer mutant. As a result, as shown in FIG. 2, the establishedCTL clones were found to be strongly stained by the tetramer-positiveand CD8-positive fractions, and to specifically recognize complexesformed between the peptides of this invention and HLA antigens. Thesignals in the tetramer-negative and CD8-negative fractions are due tofeeder cells.

Example 6 Prediction of VEGFR-2 (KDR)-Derived Peptides-2 (HLA-A*0201)

In a similar manner to Example 1, 15 types of 9-mer peptides (SEQ IDNOs:26 to 40), and 12 types of 10-mer peptides (SEQ ID NOs:41 to 52)were predicted from the entire amino acid sequence of the KDR protein,in order from highest binding affinity with HLA-A*0201, using HLAPeptide Binding Prediction software (on the World Wide Web atbimas.dcrt.nih.gov/cgi-bin/molbio/ken parker_comboform) (Table 4). Table4 shows the binding affinity of each of the 9 mers and 10 mers in orderfrom the highest value, together with the position of their N terminalin the amino acid sequence of the KDR protein. In the table, CEA588 (SEQID NO:53) refers to one of the epitopes of tumor antigen CEA(carcinoembryonic antigen) reported by Tanaka, H. et al. (posterpresentation, AACR #3669, vol. 42, p 681-682, March 2001).

TABLE 4 Binding of HLA-A*0201 to KDR-derived peptides SEQ ID INITIATIONAA SEQUENCE BINDING SEQ ID INITIATION AA SEQUENCE BINDING NO: POSITION(9 mers) AFFINITY NO: POSITION (10 mers) AFFINITY 26 KDR1093 VLLWEIFSL1792 41 KDR1094 LLWEIFSLGA 1092 27 KDR 633 SLQDQGDYV 769 42 KDR   5VLLAvALWLC 539 28 KDR   5 VLLAVALWL 739 43 KDR 313 LMTKkNSTFV 470 29 KDR775 AMFFWLLLV 427 44 KDR 779 WLLLvIILRT 292 30 KDR 773 VIAMFFWLL 270 45KDR 505 ALIEgKNKTV 285 31 KDR1034 ILLSEKNVV 179 46 KDR1084 IQSDvWSFGV285 32 KDR 604 KNLDTLWKL 128 47 KUR 733 GLYTcQACSV 223 33 KDR 772AVIAMFFWL 113 48 KDR 780 LLLViILRTV 201 34 KDR1328 KLIEIGVQT 107 49KDR   6 LLAVaLWLCV 201 35 KDR 698 IMWFKDNET 76 50 KDR 137 YITEnKNKTV 18036 KDR 608 TLWKLNATM 41 51 KDR1035 LLSEkNVVKI 167 37 KDR 491 FQGGNKIEV32 52 KDR   4 KVLLaVALWL 133 38 KDR 435 YQYGTTQTL 32 39 KDR 505ALIEGKNKT 31 40 KDR 190 YMISYAGMV 30 53 CEA 588*¹ DVLYGPDTPI 0.25*¹Tanaka H et al.: Poster presented at ACCR 2001

Example 7 Determination of HLA-A*0201 Immobilizing Epitope Peptides

CTLs were induced by a vaccination method, using the 16 types of 9-merand 10-mer peptides (SEQ ID NOs:26 to 41) predicted in Example 6 ontransgenic mice that express human HLA (A0201) (Hum. Immunol., 2000August; 61 (8):764-79 Related Articles, Books, Linkout Induction of CTLresponse by a minimal epitope vaccine in HLA A*0201/DR1 transgenic mice:dependence on HLA class II restricted T(H) response, BenMohamed, L.,Krishnan, R., Longmate, J., Auge, C., Low, L., Primus, J., and Diamond,D. J.).

Six- to eight-week old BALB/C(H-2^(d)) mice were obtained from CLEAJapan. A2/Kb transgenic mice (TGM) were provided by F. Jim Primus, Ph.D.at Vanderbilt-Ingram Cancer Center. T2 cells (TAP-deficient andHLA-A*0201-positive cells) were provided by Prof. H. Shiku at MieUniversity's Third Department of Internal Medicine.

Bone marrow was collected from mouse femur and tibia, and lymphocytesand granulocytes were removed using anti-CD4, CD8, Gr-1 antibodies(Beckton-Dickinson), anti-B220 antibody (Bioscience) and rabbitcomplement (PeL-Freez). The obtained cells were cultured in a 6-wellplate. Suspended cells were collected the following day to culture inRPMI1640 (Invitrogen) medium supplemented with 10% FBS in another 6-wellplate, to which 1,000 U/mL of mouse GM-CSF (provided by Kirin Brewery)and 1,000 U/mL of mouse IL-4 (PEPRO TECH) were added. Three days later,half of the above-mentioned medium was changed, and another two dayslater, the cells were cultured for 20 hours in 10 μg/mL of OK-432. Theresulting suspended cells were used as DCs. DCs were reacted withFITC-labeled anti-ClassII and CD40 antibodies, and PE-labeledanti-ClassI, CD11c, CD80, and CD86 antibodies (all fromBeckton-Dickinson), and confirmed to be DCs by analysis of the surfaceantigens by FACS-Calibur (Beckton-Dickinson) using Cell Quest software.

Next, 100 μg of each peptide, 140 μg of HbcAg120-140 helper peptide, and100 μL of IFA were mixed (200 μL in total). The mixture was administeredsubcutaneously to the right abdomen on day 0, and to the left abdomen onday 11. The spleens of the vaccinated mice were collected on day 21,erythrocytes were hemolytically removed using red lysis buffer (Sigma),and a portion of those cells were used as responder cells for IFN-γELISPOT assay. The remaining cells were placed into a 24-well plate(Corning) at 6×10⁶ cells/well with feeder cells at a ratio of 3:1, andstimulated again. Their cytotoxicity was measured five days later.Erythrocyte-free splenocytes, which were derived from syngeneic mice,were cultured for three days in the presence of 25 μg/mL oflipopolysaccharide (LPS) to use as feeder cells.

In this Example, the IFN-γ ELISPOT method was used to evaluate CTLinducibility according to SPOTs produced by IFN-γ producing cells.Flat-bottomed 96-well multiscreen plate MAHA S45 (Millipore) was treatedat 4° C. overnight with anti-mouse IFN-γ monoclonal antibody(Pharmingen). On the following day, the plate washed with PBS containing0.05% Tween 20, and then reacted with a blocking buffer at roomtemperature for two hours. Thereafter, peptide-pulsed T2 cells andunpulsed T2 cells were added to each well at 10⁵ cells/100 μL. Thesplenocytes of vaccinated mice were further added to each well at amaximum of 4×10⁶ cells/100 μL (200 μL in total), and cultured overnightat 37° C. The next day, each well was washed, and reacted for two hourswith biotinylated rat anti-mouse IFN-γ antibodies (Pharmingen). Afterwashing the plate thoroughly, Extravidin was added to each well andreacted at room temperature for two hours. After washing, alkalinephosphatase conjugate substrate (BIO-RAD) was added to the wells, whichwere left to stand at room temperature for five minutes. The blue spotsresulting from IFN-γ production were measured using KS ELISPOT compactrelease (Carl Zeiss).

Specific blue spots were determined as:Specific blue spot=(TISI(+)SPOT−TISI(−)SPOT)Those satisfying the following condition were considered effective:TISI(+)SPOT/TISI(−)SPOT≧2

As a result, five types of epitope peptides, SEQ ID NO:29 (amino acidinitiation position KDR775), SEQ ID NO:30 (KDR773), SEQ ID NO:33(KDR772), SEQ ID NO:34 (KDR1328), and SEQ ID NO:40 (KDR190) wereobtained by IFN-γ ELISPOT assay (Table 5).

TABLE 5 ELISPOT Analysis ELISPOT ANALYSIS × 40 (SFC) SEQ ID INITIATIONBINDING MOUSE1 MOUSE2 NO: POSITION AA SEQUENCE AFFINITY pep(+) pep(−)pep(+) pep(−) 26 KDR1093 VLLWEIFSL 1792 5 8 7 1 41 KDR1094 LLWEIFSLGA1092 0 0 0 0 27 KDR 633 SLQDQGDYV 769 0 0 0 0 28 KDR   5 VLLAVALWL 739 60 7 3 29 KDR 775 AMFFWLLLV 427 95 32 322 17 30 KDR 773 VIAMFFWLL 270 1716 193 1 31 KDR1034 ILLSEKNVV 179 0 0 0 0 32 KDR 604 KNLDFLWKL 128 0 7 00 33 KDR 772 AVIAMFFWL 113 88 19 143 31 34 KDR1328 KLIEIGVQT 107 0 0 815 35 KDR 698 IMWFKDNET 76 0 0 0 0 36 KDR 608 TLWKLNATM  41 0 0 0 0 37KDR 491 FQGCINKIEV 32 0 0 0 0 38 KDR 435 YQYGTTQTL 32 0 0 0 0 39 KDR 505ALIEGKNKT 31 0 0 0 0 40 KDR 190 YMISYAGMV 30 122 6 33 11 53 CEA 588*¹DVLYGPDTPI 0.25 229 2 230 6 *¹Tanaka H et al.: Poster presented at AACR2001

Example 8 Confirmation of In Vivo Antitumor Effect-1

Bone marrow was collected from six- to eight-week old male BALB/c mice(CLEA Japan, Inc.) with that same anchor motif as human HLA-A2402. As inExample 7, GM-CSF (Kirin Brewery) (1,000 U/mL) and IL-4 (Genzyme) (1,000U/mL) were added to the bone marrow and cultured to prepare dendriticcells. The dendritic cells were pulsed with the peptide of SEQ ID NO:5(amino acid initiation position KDR189) and then administered (1×10⁶cells/mouse) twice to nine BALB/c mice at the right lower quadrant, withan interval of seven days. Seven days later, colon cancer cell line(5×10⁵ cells), Colon 26 (Taiho Pharmaceutical), was inoculated to theright abdomens of the mice, and mice survival and tumor growth wereexamined.

First, KDR expression was analyzed by RT-PCR, as indicated in FIG. 3, toconfirm that KDR was not expressed in Colon 26 cells alone, butexpressed in the tumoral tissues of mice inoculated with Colon 26. Morespecifically, tumor vessels were confirmed to appear in tumoral tissues,and to form the tumor.

FIG. 3 shows the survival curve of Colon 26-inoculated mice. 35 daysafter tumor inoculation, two out of nine mice survived when HBSS (Hank'sBalanced Salt Solution) was administered alone (100 μL) as a control;five out of nine mice survived when inoculated with dendritic cells thatwere not pulsed with peptides; and all of the nine mice survived wheninoculated with dendritic cells pulsed with the peptide of SEQ ID NO:5.

FIG. 4 shows the effect on the growth of Colon 26-derived tumors inmice. Compared to the control, suppressive effects on tumor growth wereobserved when inoculating dendritic cells, and remarkable suppressioneffects were observed when inoculating dendritic cells pulsed with thepeptide of SEQ ID NO:5.

Example 9 Homology Searches for the Peptides of this Invention

Homology searches for the peptides of this invention (SEQ ID NO:2 (aminoacid initiation position KDR1318), SEQ ID NO:3 (KDR220), SEQ ID NO:5(KDR189), SEQ ID NO:8 (KDR169), SEQ ID NO:11 (KDR826), SEQ ID NO:30(KDR773), and SEQ ID NO:40 (KDR190)) were carried out by using the BLASTprogram (on the World Wide Web at ncbi.nlm.nih.gov/blast/blast.cgi). Nopeptides with a sequence completely identical to any one of thesepeptides could be found (Table 6). The 9-mer peptide (KDR169) of SEQ IDNO:8 capable of inducing strong CTL activity in Example 4 had only onesequence containing two mismatches (77.8% homology), and two sequencescontaining three mismatches (66.7% homology). The peptide of SEQ ID NO:5(KDR189), whose remarkable in vivo anti-tumor effect was observed inExample 8, had only one sequence containing three mismatches (66.7%homology).

TABLE 6 Homology analysis using the BLAST program (on the World Wide Webat ncbi.nlm.nih.gov/blast/blast.cgi) KDR169 KDR189 KDR826 KDR220 KDR1318KDR773 KDR190 IDENTITY (9/9) 0 0 0 0 0 0 0 IDENTITY (8/9) 0 0 3 0 0 0 0IDENTITY (7/9) 1 0 2 0 2 2 0 IDENTITY (6/9) 2 1 — 0 — 2 0

Example 10 Confirmation of In Vivo Anti-Tumor Effect-2

Bone marrow was collected from six- to eight-week old male A2/Kbtransgenic mice (TGM) expressing human HLA-A0201. As in Example 8,GM-CSF (Kirin Brewery) (1,000 U/mL) and IL-4 (Genzyme) (1,000 U/mL) wereadded to the bone marrow and then cultured to prepare dendritic cells.The dendritic cells were pulsed with the peptide of SEQ ID NO:30 (aminoacid initiation position KDR773), and administered (1×10⁶ cells/mouse)twice to the left lower quadrant of twelve A2/Kb transgenic mice, withan interval of seven days. Seven days later, B16 melanoma cells (ATCC)were inoculated (1×10⁶ cells) to the right abdomen of the mice, and micesurvival and tumor growth were examined.

FIG. 5 shows the survival rate of B16-inoculated mice. 35 days aftertumor inoculation, eight of twelve mice survived when HBSS wasadministered alone (100 μL) as a control; eleven of twelve mice survivedwhen inoculated with dendritic cells that were not pulsed with peptides;and all twelve mice survived when inoculated with dendritic cells pulsedwith the peptide of SEQ ID NO:30.

FIG. 6 shows the effect on the growth of B16 mice-derived tumors inmice. Compared to the control, suppressive effects were observed againsttumor growth when inoculating with dendritic cells, and remarkablesuppressive effects were observed on inoculating dendritic cells pulsedwith the peptide of SEQ ID NO:30.

Example 11 Cytotoxic Activity Against Cells Forcibly Expressing KDR

Using adenoviruses, KDR (see, J. Biol. Chem., 1994 Oct. 28; 269(43):26988-95) was forcedly expressed in HLA-A24-positive human coloncancer HT29 strain (ATCC), according to the method described by Miyake,S. et al., Proc. Natl. Acad. Sci. USA., 93, 1320-1324 (1996). Morespecifically, based on the titer determined by plaque assay of human 293cells, cells were infected at a specific multiplicity. HT29 cells wereplated at a cell density of 105 cells in a 60-mm dish, and incubated for24 hours. On the following day, the medium was removed and exchangedwith 200 μL of viruses continuously diluted with fresh medium. The cellswere incubated at 37° C. for one hour, and then growth medium was addedto the cells, which were cultured for 48 hours to be used as targetcells. HLA-A24-positive HT29 strain, forced by adenoviruses to expressEGFP (see, Leukemia 1999 April; 13 (4):605-13), was used as a control.HT29 strains forcedly expressing KDR and EGFP, respectively, canpotentially present partial peptides of each protein on their respectivecell surface.

Using CTL clone C29-169, induced from the peptide of SEQ ID NO:8 (aminoacid initiation position KDR169), as described in Example 3, which bindsto HLA-A24, the cytotoxic effect on HLA-A24-positive HT29 cells forcedto express KDR was measured by chromium release assay. The results areshown in FIG. 7.

Cytotoxic activity was measured by ⁵¹Cr release assay for four hours.The target cells were cultured in a 6-well plate at a concentration of1×10⁶ cells/well for 24 hours, infected with adenovirus to which KDR orEGFP has been inserted (Ad-KDR or Ad-EGFP) at MOI 50, and used after 48hours. The target cells were labeled with 100 μCi of Na₂ ⁵¹CrO₄ at 37°C. for one hour, and washed three times with RPMI1640. The target cells(1×10⁴ cells/100 μL) and 100 μL of effector cells at variousconcentrations were added (200 μL in total) to a U-shaped 96-wellmicrotiter plate (Corning), and cultured in a CO₂ incubator at 37° C.for four hours. After culturing, 100 μL of supernatant was collectedfrom each well and measured using a γ counter to calculate cytotoxicity,in the same manner as in Example 2.

As a result, as shown in FIG. 7, CTL clone C29-169 showed remarkablyhigh cytotoxic effect towards HLA-A24-positive HT29 forcedly expressingKDR, compared to that towards cells forcedly expressing EGFP.

Example 12 Cytotoxic Activity Against Cells Endogenously Expressing KDR

The cytotoxic effect of the CTL clone C29-169 described in Example 3,against HUVEC KT5 strain, derived from HLA-A24-positive human umbilicalcord vascular endothelial cells that endogenously express KDR, wasexamined using KDR-positive and HLA-A24-negative HUVEC strain, P8, as acontrol. These cells present partial peptides of KDR on their cellsurface.

Cytotoxic activity was measured by a four hour ⁵¹Cr release assay. Thetarget cells were labeled with 100 μCi of Na₂ ⁵¹CrO₄ at 37° C. for onehour, then washed three times with RPMI1640. Target cells (1×10⁴cells/100 μL) and 100 μL of effector cells at various concentrationswere added (200 μL in total) to a U-shaped 96-well microtiter plate(Corning), and cultured in a CO₂ incubator at 37° C. for four hours.After culturing, 100 μL of supernatant was collected from each well andmeasured using a γ counter to calculate the cytotoxicity in the samemanner as in Example 2.

As apparent from the results of FIG. 8, CTL clone C29-169 showedcytotoxic effect against HLA-A24-positive HUVEC endogenously expressingKDR, and showed weak cytotoxic effect against HLA-A24-negative HUVEC.

Example 13 Establishment of CTL Clones Using HLA-A0201-Binding Peptides

By using the peptide of SEQ ID NO:40 (amino acid initiation positionKDR190), KDR773-2L peptide (SEQ ID NO:54) in which the second amino acidfrom the N terminal in the peptide of SEQ ID NO:30 (KDR773) has beenconverted to leucine, the peptide of SEQ ID NO:29 (KDR775), the peptideof SEQ ID NO:34 (KDR1328), and the peptide of SEQ ID NO:33 (KDR772),which bind to HLA-A0201 and which were determined to be effectiveaccording to the ELISPOT analysis in Example 7, two types (KWC14-190 andKWC65-190), four types (KWC44-773-2L, KWC76-773-2L, KWC129-773-2L, andKWC134-773-2L), two types (KWC81-775 and KWC85-775), twelve types(KWC16, KWC21, KWC22, KWC47, KWC51, KWC108, KWC117, KWC132, KWC151,KWC153, KWC156, and KWC159), and one type (KWC72-772) of CTL clones,respectively, were obtained as in Example 3. The respectivecytotoxicities are shown in Table 7.

TABLE 7 CYTOTOXIC ACTIVITY ×20 ×2 CLONE NAME Pep(+) Pep(−−) Pep(+)Pep(−−) KWC14 -190 43% 0%  9% 0% KWC65 -190 83% 0% 27% 0% KWC44 -773 -2L87% 0% 64% 0% KWC76 -773 -2L 83% 2% 68% 1% KWC129 -773 -2L 89% 0% 82% 1%KWC134 -773 -2L 89% 2% 54% 2% KWC81 -775 86% 0% 64% 0% KWC85 -775 90% 0%63% 0% KWC16 95% 0% 85% 0% KWC21 100%  0% 91% 0% KWC22 93% 0% 77% 0%KWC47 109%  2% 87% 1% KWC51 101%  0% 93% 1% KWC108 71% 2% 23% 2% KWC11783% 0% 42% 0% KWC132 102%  0% 55% 0% KWC151 102%  0% 101%  0% KWC153 47%0% 18% 0% KWC156 64% 0% 23% 0% KWC159 93% 0% 86% 0% KWC72-772 101%  0%67% 0%

As apparent from the results in Table 7, the peptides of SEQ ID NOs:29,33, 34, and 40, as well as the peptide of SEQ ID NO:54, in which thesecond amino acid from the N terminus in the peptide of SEQ ID NO:30 isleucine, can all induce CTLs having remarkable cytotoxicity, and werethus shown to be effective as epitope peptides.

Example 14

The peptide of SEQ ID NO:29 (amino acid initiation position KDR775) wasadded to HLA-A0201-positive T2 strain to produce a target cell. By usingthe cell, the cytotoxic effect of the CTL clone (KDR C85-775(KWC85-775)) obtained from the peptide of SEQ ID NO:29 in Example 13 wasexamined by chromium release assay.

In the results, shown in FIG. 9, CTL clone C85-775 (KWC85-775) clearlyshowed a cytotoxic effect against HLA-A0201-positive cells presentingthe peptide of SEQ ID NO:29. In contrast, absolutely no cytotoxic effectwas observed against control cells not presenting the peptide of SEQ IDNO:29.

Example 15

The cytotoxic effect of the CTL clone (KDR C85-775 (KWC85-775)) againsthepatocellular carcinoma cell line HLA-A0201-positive HePG2, forced byadenoviruses to express KDR, was examined by chromium release assay.HLA-A0201-positive HePG2 forced to express EGFP was used as control.

In the results, shown in FIG. 10, CTL clone KDR C85-775 (KWC85-775)showed remarkably high cytotoxic effect against HLA-A0201-positive cellspresenting KDR peptides on the surface, compared to activity againstcells presenting the EGFP peptide.

Example 16

The peptide of SEQ ID NO:34 (amino acid initiation position KDR1328) wasadded to HLA-A0201-positive T2 strain to produce target cells. Usingthese cells, the cytotoxic effect of the CTL clone (KDR C51-1328(KWC51)) obtained from the peptide of SEQ ID NO:34 in Example 13 wasexamined by chromium release assay.

In the results, shown in FIG. 11, CTL clone KDR C51-1328 (KWC51) clearlyshowed a cytotoxic effect against HLA-A0201-positive cells presentingthe peptide of SEQ ID NO:34. In contrast, absolutely no cytotoxic effectwas observed against control cells not presenting the peptide of SEQ IDNO:34.

Example 17

The cytotoxic effect of the CTL clone (KDR C51-1328 (KWC51)) againsthepatocellular carcinoma cell line HLA-A0201-positive HePG2, forced byadenoviruses to express KDR, was examined by chromium release assay.HLA-A0201-positive HePG2 forced to expressing EGFP was used as control.

In the results, shown in FIG. 12, CTL clone KDR C51-1328 (KWC51) showedremarkably high cytotoxicity against HLA-A0201-positive cells presentingKDR peptides on the surface, compared to activity against cellspresenting the EGFP peptide.

Example 18

The peptide of SEQ ID NO:54 (KDR773-2L), in which the second peptide ofSEQ ID NO:30 is converted to leucine, was added to HLA-A0201-positive T2strain to produce target cells. Using these cells, the cytotoxic effectof the CTL clone (KDR C44-773-2L (KWC44-773-2L)) obtained from thepeptide of SEQ ID NO: 54 in Example 13 was examined by chromium releaseassay. FIG. 13 shows the results.

In the results, shown in FIG. 13, CTL clone KDR C44-773-2L(KWC44-773-2L) clearly showed a cytotoxic effect againstHLA-A0201-positive cells presenting KDR773-2L. In contrast, absolutelyno cytotoxic effect was observed against control cells not presentingKDR773-2L.

Example 19

The cytotoxic effect of the CTL clone (KDR C44-773-2L (KWC44-773-2L)obtained from the peptide of SEQ ID NO:54 (KDR773-2L), in which thesecond peptide of SEQ ID NO:30 has been converted to leucine, wasexamined by chromium release assay using HLA-A0201-positive T2 strainpulsed with the unmodified peptide of SEQ ID NO:30 (amino acidinitiation position KDR773) as the target cell.

In the results, shown in FIG. 14, CTL clones obtained by stimulationwith the modified peptide could recognize and damage thepost-modification peptide.

Example 20

The cytotoxic effect of the CTL clone (KDR C44-773-2L (KWC44-773-2L))against hepatocellular carcinoma cell line HLA-A0201-positive HePG2,forced by adenoviruses to express KDR, was examined by chromium releaseassay. HLA-A0201-positive HePG2 forced to express EGFP was used ascontrol.

In the results, shown in FIG. 15, CTL clone KDR C44-773-2L(KWC44-773-2L) showed remarkably high cytotoxic effect againstHLA-A0201-positive cells presenting KDR peptides on the surface,compared to activity against cells presenting the EGFP peptide.

Example 21

The present inventors determined whether various antibodies can blockthe cytotoxic effect of the CTL clone (KDR C51-1328 (KWC51)), obtainedfrom the peptide of SEQ ID NO:34, against hepatocellular carcinoma cellline HLA-A0201-positive HePG2, forced by adenoviruses to express KDR.

In the results, shown in FIG. 16, cytotoxic function was inhibited byanti-HLA Class I antibody against HLA antigens displayed by targetcells, and anti-CD8 antibody against CD8 used as a marker for cytotoxicT cells.

Example 22 Examination of Angiogenesis-Inhibiting Activity by Dorsal AirSac (DAS) Assay

The actual angiogenesis-inhibiting activity of peptide epitopes wasevaluated in mice using a DAS assay (see, Clinical Cancer Research, Vol.5, 2185-2191, August 1999; Cancer Research 62, 6116-6123, Nov. 1, 2002).

Specifically, a membrane filter was fixed to both sides of a chamberring, and sterilized with ethylene oxide gas. B16 melanoma cells wereprepared to 5×10⁵ cells/0.15 mL, and injected into the chamber. Mice(A2/kb transgenic mice (derived from B16)) were immobilized face down,and an air sac was produced by injecting 5 to 10 mL of air in to themurine dorsal hypoderm using a syringe. An approximately 1-cm incisionwas made to the lower part of the sac. The chamber ring, filled withcell suspension solution, was implanted in the dorsal hypoderm, and theskin was closed using a skin stapler. Six days after implantation, themouse skin was decorticated together with the chamber, fixed in anextended form, and marked with a black rubber ring to observe the newlyformed blood vessels using a microscope. Blood vessels that are newlyformed by angiogenic factors released from malignant tumor cellscharacteristically run in a zigzag manner, and are morphologicallydifferent from naturally occurring background blood vessels. Therefore,blood vessels of 3-mm or longer within the rubber ring and running in azigzag manner were determined to be newly formed blood vessels, and wereevaluated using an angiogenesis index (AI). AIs was evaluated into sixlevels, from 0 to 5 according to the number of tortuous blood vessels.All those with five or more tortuous blood vessels were determined to be5.

Vaccination was performed 14 days prior to chamber implantation, andrepeated after one week (a total of two times) by respectively injecting5×10⁴ cells of HBSS, DC alone, or peptide-pulsed DC, to the tail vein.The results are shown in FIG. 17 and in Table 8, as shown below.

TABLE 8 Effect of DAS assay INOCULATION PEPTIDE STIMULATION ANGIOGENESISINDEX (AI) P VEHICLE — (0.7 ± 0.2) HBSS — (4.5 ± 0.2) DC NONE (4.2 ±0.4) DC KDR190 (SEQ ID NO: 40) (3.6 ± 0.5) 0.3453 DC KDR772 (SEQ ID NO:33) (2.8 ± 0.5) 0.0346 DC KDR773 (SEQ ID NO: 30) (2.2 ± 0.7) 0.0034 DCKDR773-2L (SEQ ID NO: 54) (1.3 ± 0.8) 0.0001 DC KDR775 (SEQ ID NO: 29)(2.2 ± 0.5) 0.0007 DC KDR1084 (SEQ ID NO: 46) (1.7 ± 0.3) <0.0001 

FIG. 17 and Table 8 showed that the formation of tortuous blood vesselswas clearly suppressed in the groups vaccinated with DCs pulsed with thepeptides of SEQ ID NO:40 (amino acid initiation position KDR190), SEQ IDNO:33 (KDR772), SEQ ID NO:30 (KDR773), SEQ ID NO:54 (KDR773-2L), SEQ IDNO:29 (KDR775), and SEQ ID NO:46 (KDR1084), respectively, as compared tothe group to which DC was administered alone. This indicated astatistically significant angiogenesis inhibitory effect.

Wound healing and fertility in vaccinated mice were analyzed in order toobserve the adverse effects of vaccination using these epitope peptideson normal physiological angiogenesis. However, no significant adverseeffects were found in the vaccinated mice. Furthermore, using CTL clonesthat recognize KDR peptides, cytotoxicity against non-proliferative orproliferative endothelial cells was tested in vitro to examine adverseeffects in humans. As a result, these clones showed stronger activityagainst proliferative endothelial cells than against non-proliferativeendothelial cells.

Example 23

Using A2/Kb transgenic mice expressing human HLA-A0201, the peptides ofSEQ ID NO:30 (amino acid initiation position KDR773) and SEQ ID NO:34(KDR1084) were each mixed with incomplete Freund's adjuvant, thenadministered on day 3 and 13 to colon cancer strain MC38-implantedtumor-bearing mice so as to indicate changes in tumor volume.

The in vivo anti-tumor effect in a therapeutic model ofHLA-A*0201-immobilized epitope peptide (KDR773) was evaluated using thearea of the tumor, using the same vaccination method as in Example 22.The result, as apparent from FIG. 18, confirm a significant tumor growthsuppression effect by vaccination with the peptides of SEQ ID NO:30 andSEQ ID NO:34.

Example 24

The peptide of SEQ ID NO:40 (amino acid initiation position KDR190) wasadded to HLA-A0201-positive T2 strain to produce target cells. Usingthese cells, the cytotoxic effect of the CTL clone (KWC65-190) obtainedfrom the peptide of SEQ ID NO:40 in Example 13 was examined usingchromium release assay.

In the results, shown in FIG. 19, CTL clone KWC65-190 clearly showed acytotoxic effect against HLA-A0201-positive cells presenting the peptideof SEQ ID NO:40. In contrast, absolutely no cytotoxic effect wasobserved against control cells not presenting the peptide of SEQ IDNO:40.

Example 25

The peptide of SEQ ID NO:33 (amino acid initiation position KDR772) wasadded to HLA-A0201-positive T2 strain to produce target cells. Usingthese cells, the cytotoxic effect of the CTL clone (KWC72-772) obtainedfrom the peptide of SEQ ID NO:33 in Example 13 was examined using achromium release assay.

In the results, shown in FIG. 20, CTL clone KWC72-772 clearly showed acytotoxic effect against HLA-A0201-positive cells presenting the peptideof SEQ ID NO:33. In contrast, absolutely no cytotoxic effect wasobserved against control cells that did not present the peptide of SEQID NO:33.

Example 26

The cytotoxic effect of the CTL clone (KWC72-772) against hepatocellularcarcinoma cell line HLA-A0201-positive HePG2, forced by adenoviruses toexpress KDR, was examined by chromium release assay. HLA-A0201-positiveHePG2 forced to express EGFP was used as control.

In the results, as shown in FIG. 21, CTL clone KWC72-772 showedremarkably high cytotoxic effect against HLA-A0201-positive cellspresenting KDR peptides on their surface, as compared to activityagainst cells presenting the EGFP peptide.

Example 27

The peptides of SEQ ID NO:2 (amino acid initiation position KDR1318),SEQ ID NO:3 (KDR220), SEQ ID NO:5 (KDR189), and SEQ ID NO:11 (KDR826)were added to HLA-A24-positive human B-lymphocyte strain A24-LCL(HLA-A24/24, Takara Shuzo) to produce target cells. Using these cells,the cytotoxic effects of CTL clones, C7-1318, KWC46, C18-189, andC65-826, described in Example 3, were examined by chromium releaseassay.

In the results, shown in FIGS. 22 to 25, each of the CTL clones,C7-1318, KWC46, C18-189, and C65-826, clearly showed a cytotoxic effectagainst each of the HLA-A24-positive cells presenting the peptides ofSEQ ID NOs:2, 3, 5, and 11, respectively. In contrast, hardly any orabsolutely no cytotoxic effect was observed against each of the controlcells, which did not present the peptides.

Example 28

As for Example 11, the cytotoxic effects of the CTL clones, C7-1318,KWC46, C18-189, and C65-826, described in Example 3, against human coloncancer HT29 strain (ATCC), forced by adenoviruses to express KDR, wereexamined by chromium release assay. HLA-A24-positive HT29 strainforcedly expressing EGFP was used as a control.

In the results, shown in FIGS. 26 to 29, all CTL clones, C7-1318, KWC46,C18-189, and C65-826, showed remarkably high cytotoxic effect againstHLA-A24-positive HT29 forcedly expressing KDR, compared to activityagainst cells forcedly expressing EGFP.

Example 29

As for Example 22, the angiogenesis inhibitory activity of peptideepitopes against the angiogenic response induced by Colon 26 cells inBALB/c mice was evaluated by DAS assay.

As apparent from the results of FIGS. 30 and 31, in the groupsvaccinated with DCs pulsed with the peptides of SEQ ID NO:5 (amino acidinitiation position KDR189) and SEQ ID NO:11 (KDR826), respectively, theformation of tortuous blood vessels was clearly suppressed as comparedto the group administered with DC alone. This indicated a statisticallysignificant angiogenesis suppression effect.

Example 30

The CTL response against each type of following epitope peptides wasinvestigated using PBMC of cancer patients after stimulation with eachpeptide:

Peptides of SEQ ID NO:8 (amino acid initiation position KDR169), SEQ IDNO:5 (KDR189), SEQ ID NO:3 (KDR220), SEQ ID NO:11 (KDR826), and SEQ IDNO:17 (KDR1318), with high HLA-24 binding affinity; and peptides of SEQID NO:40 (KDR190), SEQ ID NO:33 (KDR772), SEQ ID NO:30 (KDR773), SEQ IDNO:29 (KDR775), and SEQ ID NO:34 (KDR1328), with high HLA-A0201 bindingaffinity.

Using the method reported by Maeda, Y. et al., Br. J. Cancer, 87:796-804(2002), peptide-specific CTLs were detected in PBMC. First, PBMCcollected from patients (1×10⁵ cells) were incubated with 10 μM of eachpeptide in a U-shaped bottom 96-well plate comprising 200 μL medium. Themedium consisted of 45% RPMI1640 medium, 45% AIM-V medium, 10% FBS, 100U/mL interleukin-2 (IL-2), and 0.1 μM MEM non-essential amino acidsolution. Every three days, half of the medium was removed and replacedwith fresh medium containing the corresponding peptide (20 μM). Afterincubation for twelve days, cells were collected to test IFN-γproduction ability in response to each peptide. When the amount of IFN-γproduced by peptide-stimulated PBMC in response to a correspondingpeptide was two times or more than the amount produced in response tothe control HIV peptide, that test peptide was deemed positive.

These results showed that a CTL precursor is produced in HLA-A2- andHLA-A24-type cancer patients, as in a healthy donor (Table 9). In thetable, patients A, B, and C are HLA-A24-type patients; and patient D isan HLA-A02-type patient. Patients A and D are colon cancer patients; andpatients B and C are melanoma patients. CMVs refer to thecytomegalovirus-derived peptides used as positive controls. The peptideswith high A-24 binding affinity are described in Kuzushima, K. et al.,Blood 2001, 98 (6):p 1872-1881, and such; and the peptides with high A-2binding affinity are described in Solache, A. et al., J. Immunol. 1999,163 (10); p 5512-5518, and such.

TABLE 9 Cancer patient-derived CTL precursor PATIENT A PATIENT B PATIENTC PATIENT D POSITIVE TOTAL POSITIVE TOTAL POSITIVE TOTAL POSITIVE TOTALWELLS WELLS WELLS WELLS WELLS WELLS WELLS WELLS CMV 1 32 1 24 0 16 CMV 520 KDR 169 12 32 4 32 1 24 KDR 190 3 24 KDR 189 3 32 1 32 0 24 KDR 772 324 KDR 220 0 32 3 32 0 24 KDR 773 1 24 KDR 826 11 32 3 32 0 24 KDR 775 224 KDR 1318 0 32 9 32 1 24 KDR 1328 0 24

As for Example 14 and the like, a standard ⁵¹Cr release assay wasperformed for four hours to establish CTL lines from the CTL precursors(FIG. 32).

INDUSTRIAL APPLICABILITY

The present invention provides novel peptides, which induce cytotoxic Tcells by targeting the endothelial cells formed in a wide range oftumoral tissues, and which are extremely effective as cancer vaccines.The present invention also provides pharmaceuticals for treating andpreventing tumors, where the pharmaceuticals comprise these peptides.

All publications, patents, and patent applications cited herein areincorporated into the present description by reference.

1. A nonapeptide consisting of the amino acid sequence of SEQ ID NO:8.2. A peptide with cytotoxic T cell inducibility, wherein one or twoamino acids are substituted or added to the amino acid sequence of SEQID NO:8.
 3. The peptide of claim 2, wherein the second amino acid fromthe N terminus is phenylalanine, tyrosine, methionine, or tryptophan. 4.The peptide of claim 2, wherein the C terminal amino acid isphenylalanine, leucine, isoleucine, tryptophan, or methionine.
 5. Thepeptide of claim 3, wherein the C terminal amino acid is phenylalanine,leucine, isoleucine, tryptophan, or methionine.
 6. A nonapeptideselected from the peptides consisting of the amino acid sequence of SEQID NO:2, 3, 5, 11, and
 12. 7. A peptide with cytotoxic T cellinducibility, wherein one or two amino acids are substituted or added tothe amino acid sequence of SEQ ID NO:2, 3, 5, 11, or
 12. 8. The peptideof claim 7, wherein the second amino acid from the N terminus isphenylalanine, tyrosine, methionine, or tryptophan.
 9. The peptide ofclaim 7, wherein the C terminal amino acid is phenylalanine, leucine,isoleucine, tryptophan, or methionine.
 10. The peptide of claim 8,wherein the C terminal amino acid is phenylalanine, leucine, isoleucine,tryptophan, or methionine.
 11. A pharmaceutical composition comprisingone or more peptides of any one of claims 1 or
 6. 12. A method forinducing in vitro an antigen-presenting cell with high cytotoxic T cellinducibility the method comprises contacting said cell with any one ofthe peptides of claim
 6. 13. A method for inducing in vitro a cytotoxicT cell the method comprises contacting said cell with any one of thepeptides of claim
 6. 14. An isolated cytotoxic T cell that is producedby contacting said cell with any one of the peptides of claim 1 or 6.15. An isolated antigen-presenting cell that is produced by contactingsaid cell with any one of the peptides of claim 1 or
 6. 16. Acomposition for inhibiting angiogenesis at a diseased site, wherein thecomposition comprises the peptide of claim 1 as an active ingredient.17. The composition of claim 16, which is used for administration to asubject whose HLA antigen is HLA-A24.
 18. The composition of claim 16,which is used to suppress the growth and/or metastasis of malignanttumors.
 19. The composition of claim 17, which is used to suppress thegrowth and/or metastasis of malignant tumors.
 20. A composition forinhibiting angiogenesis at a diseased site, wherein the compositioncomprises any one of the peptide of claim 6 as an active ingredient. 21.The composition of claim 20, which is used for administration to asubject whose HLA antigen is HLA-A24.
 22. The composition of claim 20,which is used to suppress the growth and/or metastasis of malignanttumors.
 23. The composition of claim 21, which is used to suppress thegrowth and/or metastasis of malignant tumors.